effect of cocoa polyphenolic extract on macrophage...

Research ArticleEffect of Cocoa Polyphenolic Extract on MacrophagePolarization from Proinflammatory M1 to Anti-InflammatoryM2 State

Laura Dugo,1 Maria Giovanna Belluomo,1 Chiara Fanali,1 Marina Russo,1

Francesco Cacciola,2 Mauro Maccarrone,1,3 and Anna Maria Sardanelli1,4

1Department of Medicine, Campus Bio-Medico University of Rome, Via A. del Portillo 21, 00128 Roma, Italy2Department of Biomedical, Dental Sciences and Morphological and Functional Images, University of Messina, Via ConsolareValeria, 98125 Messina, Italy3European Center for Brain Research, Santa Lucia Foundation IRCCS, Rome, Italy4Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari “Aldo Moro”, P.zza G. Cesare, 11,70124 Bari, Italy

Correspondence should be addressed to Anna Maria Sardanelli; [email protected]

Received 23 September 2016; Revised 22 November 2016; Accepted 20 April 2017; Published 28 June 2017

Academic Editor: Chung-Yen Oliver Chen

Copyright © 2017 Laura Dugo et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Polyphenols-rich cocoa has many beneficial effects on human health, such as anti-inflammatory effects. Macrophages function ascontrol switches of the immune system, maintaining the balance between pro- and anti-inflammatory activities. We investigatedthe hypothesis that cocoa polyphenol extract may affect macrophage proinflammatory phenotype M1 by favoring an alternativeM2 anti-inflammatory state on macrophages deriving from THP-1 cells. Chemical composition, total phenolic content, andantioxidant capacity of cocoa polyphenols extracted from roasted cocoa beans were determined. THP-1 cells were activated withboth lipopolysaccharides and interferon-γ for M1 or with IL-4 for M2 switch, and specific cytokines were quantified. Cellularmetabolism, through mitochondrial oxygen consumption, and ATP levels were evaluated. Here, we will show that cocoapolyphenolic extract attenuated in vitro inflammation decreasing M1 macrophage response as demonstrated by a significantlylowered secretion of proinflammatory cytokines. Moreover, treatment of M1 macrophages with cocoa polyphenols influencesmacrophage metabolism by promoting oxidative pathways, thus leading to a significant increase in O2 consumption bymitochondrial complexes as well as a higher production of ATP through oxidative phosphorylation. In conclusion, cocoapolyphenolic extract suppresses inflammation mediated by M1 phenotype and influences macrophage metabolism by promotingoxidative pathways and M2 polarization of active macrophages.

1. Introduction

Monocyte-derived macrophages play a crucial role in inflam-mation, host defense, and tissue repair [1, 2]. Macrophageshave important pathogenic roles in many chronic diseases,such as asthma, inflammatory bowel disease, atherosclerosis,rheumatoid arthritis, and fibrosis [2–4]. Many studies haveattempted to simulate the process of monocyte to macro-phage differentiation in vitro, through the culture of mono-cytes with or without addition of different cytokines [5].Phenotypic and functional flexibility is a key property of

macrophages [6–9]. In vivo, human macrophages can beclassified into two categories according to their activationstates. A classical M1 phenotype and an alternative M2 phe-notype have been characterized on the basis of their reactionsto different stimuli. Over the last few years, considerableprogress has been made toward characterization of epige-netic mechanisms, transcription, and posttranscriptional fac-tors regulating macrophage polarization [10]. M1 activationis drived by the cytokine interferon- (IFN-) γ and the activa-tion of Toll-like receptors (TLRs) by lipopolysaccharide(LPS), while M2 activation is triggered by interleukin- (IL-)

HindawiOxidative Medicine and Cellular LongevityVolume 2017, Article ID 6293740, 11 pageshttps://doi.org/10.1155/2017/6293740

https://doi.org/10.1155/2017/6293740

4 [6]. It is now known that other mediators, besides IL-4, aswell as IL-13 can drive M2 polarization [11, 12]. However,M1 and M2 phenotypes are two limits of a variety of func-tional states, which make up the complexity of macrophageflexibility [13–15]. M1 macrophages are characterized byhigh production of proinflammatory cytokines (includingtumor necrosis factor- (TNF-) α, IL-1β, IL-6, and IL-12)and reactive nitrogen and oxygen species (RNS, ROS),promoting T helper- (h-) 1 cells that express IFN-γresponse and having strong tumoricide and microbicideactivity [15]. On the other hand, M2 macrophages haveimmune regulatory function, an efficient phagocytic activityand are characterized by high levels of scavenging molecules,by the production of ornithine and polyamines throughthe arginase pathway and by the expression of mannoseand galactose receptors [9].

The onset of a pathological state is frequently associatedwith dynamic changes in macrophage stimulation; in fact,M1 macrophages are involved in initiating and sustaininginflammation while M2 macrophages are linked with resolu-tion or chronic inflammation [7, 16]. Polarized phenotypesare reversible in vitro and in vivo [17–19]. Modulationof macrophage function is an off-target effect for varioustherapeutic agents such as STAT3/STAT6 inhibitors, imi-dazoquinolines, peroxisome proliferator-activated receptor-(PPAR-) γ agonists, and CD40 [6].

Bioactive food compounds, such as the polyphenols, haverecently gained consideration for their anti-inflammatoryproperties, which might have an impact on macrophagephenotype favoring an alternative M2 anti-inflammatorystate. It was demonstrated that pomegranate polyphenolsdose dependently attenuated macrophage response to M1proinflammatory activation in the J774.A1 macrophage-likecell line [20]. This was supported by a significant decreasein the proinflammatory cytokine secretion in response tostimulation by IFN-γ and LPS and by a significant increasein IL-10 secretion promoting the differentiation of a M2anti-inflammatory phenotype [20].

Various in vitro studies have attributed downregulationof the inflammatory response to cocoa polyphenols. Cocoa,a product derived from the beans of Theobroma cacao plant,is a rich source of monomeric polyphenolic antioxidants,mainly epicatechin and catechin, and various polymersderived from these monomers, identified as procyanidins[21]. Cocoa has a potent antioxidant capacity, as comparedwith other products [22], related to flavonoid content[23]. In addition to their potent antioxidant characteristic,cocoa polyphenols were shown to possess remarkable anti-inflammatory properties [24]. In vitro, flavonoids presentin cocoa decreased the production of inflammatory cyto-kines (TNF-α, IL-6, and IL-1β), ROS, and RNS, in LPS-stimulated macrophages [25]. In a healthy population ofSouthern Italy, regular intake of dark chocolate was inverselyassociated to serum C-reactive protein level [26, 27]. Fur-thermore, cocoa treatment was shown to be effective in theprevention of cardiovascular diseases and in the modulationof blood pressure in animal and human studies [20–30].With regard to its effect in inflammatory conditions, they stillneed to be further explored [31]. In animal models of

diseases, such as inflammatory bowel disease, arthritis, andcolitis, cocoa polyphenols reduced both the inflammatoryresponse and the oxidative stress [32, 33]. In this context,cocoa has been shown to modulate the immune system, inparticular, the systemic and intestinal immune responsesand the inflammatory innate response [34].

About the mechanism by which cocoa flavonoids modu-late immune function, it has been suggested that they reduceredox sensitive nuclear factor kappa-light-chain-enhancer ofactivated B cell (NF-kB) activation and, consequently, theexpression of many genes involved in cytokine secretion[24, 35, 36]. They can also directly interact with gene expres-sion factors and cell signaling involved in cytokine secretion,such as activator protein 1 (AP-1) [37] and signal transducerand activator of transcription 4 (STAT4) [38]. Furtherstudies are needed to elucidate the interactions betweencocoa and the redox-sensitive signalling pathways involvedin the expression of many genes and, consequently, in severalcell functions, such as the immune response.

The identification of mechanisms and molecules asso-ciated with macrophage flexibility and polarization willprovide a basis for macrophage-centered diagnostic andtherapeutic strategies. Although previous studies demon-strated that cocoa polyphenols possess anti-inflammatoryeffects, to the best of our knowledge, a research focusedon the direct effect of the cocoa polyphenols on macro-phage inflammatory phenotype has never been performed.

In this study, we have investigated the hypothesis that acocoa polyphenolic extract may influence the macrophagepolarization through the metabolic switch promoting a M2anti-inflammatory state.

2. Materials and Methods

2.1. Cocoa Polyphenol Extraction. Cocoa beans, commer-cially available locally, was originated from Ghana, WestAfrica. Polyphenol extraction was carried out as previouslydescribed [26] with some modifications as shown below.To remove lipids, cocoa beans (3 g) were ground to a pow-der with quartz sand, then was vortexed in 10ml of hex-ane, and centrifuged for 5 minutes at 800×g and 4°C.Hexane extraction was repeated for three times. Subse-quently, nonfat cocoa grain was dissolved in 10ml ofmethanol/water solution, 70 : 30 (v/v), and polyphenolswere extracted by three centrifugations at 800×g and 4°Cfor 5 minutes. Cocoa polyphenolic extract was filtered andevaporated until dry in a rotary evaporator. Finally, sampleswere dissolved in methanol/water/acetic acid solution,70 : 28 : 2 (v/v/v).

2.2. Determination of Phenolic Compounds and TotalAntioxidant Capacity in Cocoa Polyphenolic Extract. Pheno-lic content of cocoa extract was measured by a modifiedFolin-Ciocalteu method using gallic acid as standard [39].The diluted 1 : 40 (v/v) aqueous solution of extract (20μl)was mixed with Folin-Ciocalteu reagent (100μl) and wasincubated with Na2CO3 1.89M for 2 hours. The absorbancewas measured at 765nm by a multifunctional microplatereader (InfiniteM 200 Pro, TECAN, Italy) in the samples

2 Oxidative Medicine and Cellular Longevity

dispensed in triplicate in a 96-well cell culture plate (GreinerBio-One, Germany). The results were expressed as mg ofgallic acid equivalent (GAE)/l.

Total antioxidant capacity of cocoa extract was measuredby using the Trolox equivalent antioxidant capacity (TEAC)assay [40]. Briefly, 10μl of cocoa polyphenolic extract wasmixed with 200μl of ABTS+ solution in a 96-well cell cultureplate and the absorbance was recorded at 734 nm for 90seconds by a multifunctional microplate reader (InfiniteM

200 Pro, TECAN, Italy). TEAC values were calculated fromthe Trolox standard curve (60–300μM).

2.3. RP-HPLC/PDA/MS Analysis of Cocoa PolyphenolicExtract. Cocoa polyphenolic extracts, before the RP-HPLC/PDA/MS analysis, were filtered to a 0.45μm Acrodisc filter(Pall Life Sciences, Ann Arbor, MI, USA).

HPLC analyses, performed on 2μl of the injectionvolume, were carried out as previously described [40]. Inparticular, the mobile phase contained water (Sigma-Aldrich) /formic acid (Riedel-de Haën, Hanover, Germany)(99.9 : 0.1, (v/v)) (solvent A) and acetonitrile (Sigma-Aldrich)/formic acid (99.9 : 0.1, (v/v)) (solvent B), and thestepwise gradient profile was as follows: 0 minutes, 5% B;30 minutes, 30% B; 35 minutes, 100% B; and 36 minutes,0% B. Flow rate was 0.7ml/minute. An SPD-M20A UVdetector and an LCMS-2020 were employed. Data wereobtained by a photodiode array (PDA) detector in the range190–400nm. Time constant was 0.64 s and sample frequency1.5625Hz. MS acquisition was carried out using ESI interface(Shimadzu), in negative mode by the LCMS solution Ver.3.30 software (Shimadzu). 200μl of the whole LC flow ratewas directed to the interface, and the total flow was switchedto waste (500μl) and to interface (200μl) by means of a flowsplitter. ESI conditions: mass spectral range, m/z 100–1200;even time, 1 s; scan speed, 1154 amu/s; nebulizing gas (N2)flow, 1.5 l/minute; ESI temperature, 300°C; heat block,300°C; DL (desolvation line) temperature, 250°C; DL voltage,34V; probe voltage, +4.5 kV; Qarray voltage, 1.0V; anddetection gain, 1.05 kV.

2.4. Cell Culture and Differentiation. The THP1 cell line wasacquired from ATCC and cultivated in RPMI 1640 medium(Lonza, Belgium) supplemented with 10% fetal bovine serum(FBS; Lonza, Belgium), 1mM L-glutamine (Sigma-Aldrich,Milan, Italy), 100U/ml penicillin, and 100μg/ml strepto-mycin (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 37°Cto give a final concentration of approximately 2× 105cells/ml.THP-1 cellswere differentiated for 72hours intomacrophages(M0) by stimulation with 100ng/ml phorbol 12-myristate13-acetate (PMA, Sigma-Aldrich, Milan, Italy). After mac-rophage differentiation, cells were cultured for another 24hours with either LPS (1μg/ml) + INF-γ 20 ng/ml to gener-ate M1-macrophages, or with IL-4 (20 ng/ml) to generateM2-macrophages [41], in the absence or in the presenceof cocoa polyphenolic extract at different concentrationexpressed as μM GAE as indicated in the figures.

2.5. Cell Viability.M1-polarized cells were seeded in a multi-wall plate at concentration of 80000 cells/well and incubated

for 24 hours in the absence (control) and in the presence ofcocoa polyphenolic extract (0.1–100 μΜ GAE). Successively,cell culture medium was discarded, and each well was washedwith 200μl Hank’s balanced salt solution (HBSS+, Gibco,Waltham, MA, USA). MTT solution (0.5mg/ml, 100μlSigma-Aldrich, Milan, Italy) was added to cells in each well,and the plate was incubated at 37°C+5% CO2 for about 3hours, until MTT formazan crystals were visible in the cul-ture liquid. Then MTT solution was removed, and dimethylsulfoxide (DMSO, Sigma-Aldrich, Milan, Italy) (70μl/well)was added to each well for dissolving the formazan crystals.Optical density (OD) was measured at 565nm using a multi-functional microplate reader (InfiniteM 200 Pro, TECAN,Italy). Viability was calculated as the ratio of the mean ofOD obtained for each condition to that of control (absenceof cocoa polyphenolic extract) condition.

2.6. Cytokine Secretion. The levels of cell-released TNF-α,IL-6, IL-1β, IL-12, and IL-10 were measured in the har-vested supernatants by using an ELISA Ready-Set-Go kit(eBioscience, San Diego, CA, USA), following the manufac-turer’s instructions. Optical density was determined usingthe microplate reader InfiniteM 200 Pro, TECAN (Italy).

2.7. Evaluation of Mitochondrial Respiratory Activity. Mito-chondrial oxygen consumption was measured polarographi-cally with a Clark-type oxygen electrode in a thermostatedgas-tight chamber (Hansatech Instruments, Norfolk, UK)[42, 43]. Measurements of substrate-supported respirationwere carried out in intact exponentially growing cells. Theculture medium was changed 1 day before the assays. Cellswere trypsinized, centrifuged, and resuspended at 2× 106cells/ml in 0.137M NaCl, 5mM KCl, 0.7mM Na2HPO4,and 25mM Tris–HCl, pH7.4. An aliquot of cell suspensionwas used for counting and protein determination. The cellswere then transferred into the polarographic chamber. Forthe measurement of respiration rates by exogenous sub-strates, after full uncoupling of the endogenous respirationof intact cells with 30μM dinitrophenol (DNP), digitonin(30μg/106 cells) was added. After 2 minutes, added respira-tory substrates were as follows: pyruvate (5mM)/malate(2.5mM) for complex I, succinate (5mM) for complex II+ complex III, in the presence of inhibitor of complex I, androtenone (200 nM) and inhibitor of complex III antimycinA (13 nM). All amounts of oxygen depletion were normalizedto cellular protein content.

2.8. ATP Measurement. Cellular ATP levels under basalconditions were measured by a luminometer (InfiniteM

200 Pro, TECAN, Italy) with the ATP lite kit (PerkinElmer, Waltham, MA) through a luciferin-luciferase reac-tion system, according to the manufacturer’s instructions.Macrophage differentiated from THP-1 cells M0, andM1/M2 polarized cells were collected from Petri disheswith 0.05% trypsin, 0.02% EDTA, pelleted by centrifuga-tion at 500×g, and washed in phosphate-buffered saline(PBS), pH7.4. For each assay, 60000 cells in multiwellplate were used. For the evaluation of ATP content understrict glycolytic conditions, M0, M1, and M2 were incubated

3Oxidative Medicine and Cellular Longevity

for 5 hours at 37°C in both in the presence of rotenone(1mmol/l) that of antimycin A (1mmol/l), [44]. An ATP cal-ibration curve was made using known concentration ATPsolutions. An aliquot of cell lysate was employed for proteincontent quantification.

2.9. Statistical Analysis. In order to evaluate the dose-response effects, we applied the one-way repeated measuresanalysis of variance, whereas the differences between treatedand untreated M0, M1, and M2 cells were analyzed by thetwo-way repeated measures ANOVA (two-factor repetition).Student-Newman-Keuls Method was used as all pairwisemultiple comparison procedure. The results are given asmean± SD. Values of p ≤ 0 05 were chosen as the criteriafor statistically significant difference.

3. Results

3.1. Cocoa Polyphenol Extract Characterization: TotalPhenolic Content, Antioxidant Capacity, and ChemicalComposition. The total phenolic content and the antioxidantcapacity of cocoa polyphenolic extract were determined byFolin-Ciocalteu and TEAC assays, respectively. The resultspresented in Table 1 suggest that the values of total phenoliccontent, expressed as mg gallic acid equivalent (GAE)/l,

reflect the antioxidant activity, expressed as mM Troloxequivalent (TE)/g fresh mass, of cocoa extract.

Through HPLC-PDA-MS qualitative analysis, chemicalcomposition of cocoa polyphenol extract was determined(Figure 1, Table 2). As can be seen from the chromatogramreported in Figure 1, the sample analyzed contains 23 bioac-tive molecules. Phenolic acids, flavan-3-ols, alcaloids, andcyanidins represent the class of bioactive molecules presentin the sample of our interest.

3.2. Cocoa Polyphenols Increased Viability and AttenuateMacrophage Inflammatory Response. To test the effect ofcocoa polyphenols on cell viability and inflammatoryresponse, THP-1 macrophages were incubated with increas-ing concentration of CPE, expressed as μΜ GAE (0.1–100μΜ GAE), earlier to exposure to M1 activation induced bythe INF-γ and LPS.

As presented in Figure 2(a), cocoa extract at up to 100μΜ GAE concentration failed to display toxicity toward dif-ferentiated cells. Indeed, after exposure to higher concentra-tions of cocoa extract from 5 μΜ up to 100 μΜ GAE for 24hours, cell viability was significantly increased. In particular,cocoa extract at 100 μΜ GAE increased cell viability of about30% compared to control (Figure 2(a)).

Table 1: Chemical properties of cocoa polyphenol extract. GAE, gallic acid equivalent, TE, Trolox equivalent. Data were expressed as mean offive replicates± standard deviation. Polyphenol extraction yield was calculated as follows: % (w/w) =mass in dry basis/mass of initial weightfed for extraction× 100.

Beans weight(g)

Extraction solvent(v/v)

Yield (mg polyphenols/gfresh mass)

Polyphenol content(mg GAE/l)

Antioxidant capacity(mM TE/g fresh mass)

3 Me OH/ H2O (70 : 20) 170 8.4± 0.5 158± 1.6

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min0

255075

100125150175200225250275300325350375

mAU280 nm, 4 nm (100)

1

2

4

56

8

910

11

12

13

14

16

17

18

19 2021

2322

3

15

Figure 1: RP-HPLC-PDA chromatogram (extracted at 280 nm) of cocoa polyphenol extract. The chromatogram shows the phenoliccompounds present in the cocoa extract. For peak identification, see Table 2. Almost 18 compounds were identified by LC-MS as describedunder the Materials andMethods.


Later, for investigating the effect of cocoa polyphenolicextract on inflammatory response, production of proinflam-matory cytokines, IL-1β (Figure 2(b)) and IL-6 (Figure 2(c)),was quantified in M1 inflammatory macrophages in a dose-dependent manner. A concentration range from 5 μΜ upto 100 μΜ GAE was selected because it showed a significantincrease in cell viability (Figure 2(a)). The results showed inFigures 2(b) and 2(c) suggest that cocoa extract attenuatedmacrophage response to M1 proinflammatory activationthrough the reduction of about 30% (at 100 μΜ GAE) ofproinflammatory cytokines.

3.3. Cocoa Extract Influences a Macrophage M1 to M2Phenotypic Switch. To investigate the effect of cocoapolyphenolic extract (CPE) on macrophage alternative M2phenotype, the secretion of the proinflammatory and anti-inflammatory cytokines was then determined and comparedto the control basal level in untreated cells. CPE significantlydecreased macrophage response to M1 activation throughthe reduction of the secretion of proinflammatory cytokinesTNF-α (Figure 3(a)) and IL-12 (Figure 3(b)) in M1 pheno-type by up to 20% and 30%, respectively, compared tountreated cells. Furthermore, CPE increased by up 47% therelease of the anti-inflammatory IL-10 (Figure 3(c)) in M1macrophages. Cocoa extract did not exert any effect on cyto-kine secretion in M0 and M2 macrophages. Taken together,these results indicate that cocoa extract not only reduces

inflammation in M1 cells but also promotes macrophagepolarization toward the M2 alternative phenotype.

3.4. Effect of Cocoa Polyphenol Extract on M1/M2 Phenotypethrough Metabolic Switch. Macrophage metabolism wasevaluated through measurement of cellular ATP levels(Figure 4) and of oxygen consumption by mitochondrialcomplexes (Figure 5) in the differentiated THP-1 (M0) andpolarized M1 and M2 cells. The results presented inFigure 4 represent the ATP levels under strict glycolytic con-dition, in the presence of mitochondrial respiratory chaininhibitors as described under the Materials and Methods.

The results presented in Figure 4(a) show that ATP levelsdid not decrease in M1 macrophages, in the presence ofrotenone and antimycin A. On the other hand, in M0 andM2 cells, they were significantly reduced in the presence ofmitochondrial inhibitors. In Figure 4(b), the effect of cocoapolyphenolic extract on ATP levels was estimated in M0,M1, and M2. The results presented in Figure 4(b) indicatethat, after cocoa polyphenolic extract treatment, ATP levelsdecreased in M1 macrophages, in the presence of rotenoneand antimycin almost at the same levels of M0 andM1. How-ever, cocoa extract did not influence ATP levels in M0 andM2 macrophages in comparison to untreated cells.

Then, measurement of oxygen consumption wasperformed for evaluating mitochondrial functionality inmacrophage phenotypes. The activities of complex I

Table 2: Identification by HPLC-PDA-MS of cocoa polyphenolic bioactive molecule extract. UVmaximum absorption (UV), retention times(tR), and m/z ([M-H]−) values of polyphenols identified in cocoa extract were analyzed.

Peak number Compound UV tR m/z [M-H]−1 Unknown 272 1.7

2 Caffein 272 2.0 195

3 Unknown 210, 270 2.2 387, 453

4 Chrysophanol-hexoside 210, 274 3.3 415, 253

5 Quinic acid 298, 320 3.6 191

6 Vanillic acid derivative 220, 275 4.7 282

7 Unknown 296 5.1 449

8 Theobromina 272 7.5 181

9 Protocatechuic acid 228, 260, 294 8.0 153

10 Unknown 467 9.2 467

11 Cinnamic acid derivative 213, 301, 320 11.2 294

12 Catechin-3-O-glucoside 278 11.4 451, 289

13 Catechin derivate 278 12.0 497, 451

14 Catechin sulphonic acid 274 12.6 369, 289

15 Unknown 294, 298, 307 13.6 407, 305

16 Procyanidin B dimer 278 14.4 577


18 Procyanidin trimer 278 17 865

19 Procyanidin tetramer 278 17.5 1153

20 Clovamide 320 17.9 358

21 Catechin derivative 278 18.6 720


23 Dideoxyclovamide 320 23.8 326, 282


(Figure 5(a)), rotenone sensitive, and complexes II + III(Figure 5(b)), antimycin A sensitive, expressed as nmolO2/mg total proteins, were lower in the M1 macrophagesthan in M2 cells. The incubation in the presence ofcocoa polyphenol extract induced a significant increase ofoxygen consumption in M1 by both mitochondrialcomplexes (Figures 5(a) and 5(b)), thus, suggesting amore oxidative metabolism like phenotype M2. Cocoatreatment had no effect on M0 and M2 macrophages.

4. Discussion

This study demonstrates novel findings on the anti-inflammatory role of cocoa and its polyphenols, in vitro ina model of THP-1-derived macrophages. We show, for thefirst time, that cocoa extract dramatically inhibited the secre-tion of the proinflammatory cytokines TNF-α, IL-6, IL-1β,and IL-12 in INF-γ/LPS-stimulated macrophages of the same

percentage by which it increased cell viability (about 30%),compared to control. More interesting, however, is thefinding related to cocoa polyphenol-induced productionof the anti-inflammatory cytokine IL-10 in inflammatoryM1 macrophages whose levels are similar to those observedin M2 state. Thus, after cocoa treatment, M1 macrophagesshowed the same levels of IL-12 and IL-10 present in M2phenotype. In conclusion, following cocoa treatment, M1macrophages are comparable to M2 cells regarding cytokineproduction, suggesting that cocoa could promote a shifttoward an alternative M2 macrophage phenotype. Ourresults also demonstrated that cocoa extract influencesmacrophage metabolism, increasing ATP productionthrough oxidative phosphorylation and O2 consumptionby mitochondrial respiratory complexes in M1 macro-phages. ATP production by oxidative phosphorylation isblocked at the level of NADH dehydrogenase or complexI, which is the first protein in the electron transport chain,

0

20

40

60

80

100

120

140

160

0 0,1 1 5 25 50 100

�휇M GAE

Cell

viab

ility

(% ct

rl)

⁎⁎⁎

‡‡

††

⁎⁎⁎

⁎⁎⁎

†⁎⁎⁎

(a)

0

10

20

30

40

50

60

70

80

�휇M GAE

IL-1�훽

(pg/

ml)

†⁎⁎⁎

†⁎⁎⁎

⁎⁎⁎

0 5 25 50 100

†

⁎⁎⁎

(b)

0

5

10

15

20

25

30

35

40

�휇M GAE

IL-6

(pg/

ml)

†† ⁎⁎⁎

‡⁎⁎⁎

⁎⁎⁎⁎⁎⁎

0 5 25 50 100

(c)

Figure 2: Effect of cocoa polyphenolic extract on cell viability and on macrophage M1 inflammatory state. THP-1 macrophages werestimulated with 20 ng/ml INF-γ+ 100 ng/ml LPS as described under the Materials and Methods and incubated in the presence of cocoapolyphenolic extract expressed (CPE) in μΜ GAE as indicated. (a) Cell viability assayed by MTT methods was expressed as % of control(0 μΜ GAE). (b) Levels of IL-1β and (c) IL-6, determined by ELISA assay, were expressed as pg standard/ml. White bars indicate thecontrols, and grey scale the increasing concentrations of GAE. All data represent the means of 3/5 replicates± standard deviation. One-way repeated measures analysis of variance, followed by Student-Newman-Keuls method. ∗∗∗p < 0 001 cells treated with GAE versusuntreated cells; ‡p < 0 01 and †p < 0 001 other concentrations versus 100 μΜ GAE.


by rotenone and at the level of cytochrome c reductase orcomplex III, which represents the second protonic pumpby antimycin A. Both complexes contribute to generateelectrochemical gradient across mitochondrial membraneused for the synthesis of ATP by oxidative phosphoryla-tion. In M1 macrophages, in the presence of rotenoneand antimycin A, ATP levels do not changed, so M1 phe-notype has mainly a glycolytic metabolism as confirmedby others [16]. On the other hand, ATP levels are reducedin M2 cells in the presence of mitochondrial inhibitors ina significant fashion, thus, suggesting that an aliquot of

cellular ATP has been produced by oxidative phosphoryla-tion and, therefore, M2 phenotype exhibits a more oxida-tive metabolism. Once identified M1 and M2 glycolytic/oxidative metabolism, M1 macrophages produced moreATP through oxidative phosphorylation after cocoa poly-phenol treatment. This reduction of glycolytic ATP inM1 cells suggests that this phenotype acquires a moreoxidative metabolism in the presence of cocoa polyphe-nols, similar to that of M2 phenotype. However, cocoaextract does not influence ATP levels both in M0 andM2 macrophages.

⁎⁎⁎†

†

† †

0

50

100

150

200

250

300

M0 M1 M2TN

F-�훼 (

pg/m

l)CPE – – – +++

(a)

⁎⁎⁎

‡

†

†‡

0

10

20

30

40

50

M0 M1 M2

IL-1

2 (p

g/m

l)

CPE – – – ++ +

(b)

⁎⁎⁎

‡

†

†‡⁎

0

10

20

30

40

50

60

70

IL-1

0 (p

g/m

l)

CPE – – – +++M0 M1 M2

(c)

Figure 3: Effect of cocoa polyphenolic extract on M1/M2 phenotype switch. Macrophage-differentiated THP-1 (M0) cells and polarized(M1, M2) cells as described under the Materials and Methods were incubated in the absence (−) and in the presence (+) of cocoapolyphenolic extract (CPE) which concentration was expressed as 100 μM GAE. Levels of (a) TNF-α, (b) IL-12, and (c) IL-10expressed as pg/ml were measured in the collected incubation medium. Error bars represent data from 3 independent experiments:two-way repeated measures ANOVA (two-factor repetition), followed by Student-Newman-Keuls method. ∗p < 0 05, ∗∗∗p < 0 001,cells treated with CPE versus untreated. Comparisons M1 versus M2 within treatment: ‡p < 0 01 and †p < 0 001.


0

1

2

3

4

5

M0 M1 M2

nmol

O2/m

g pr

ot.

CPE – – – +++

†

⁎⁎⁎ †

††

(a)

0

1

2

3

4

5

M0 M1 M2

nmol

O2/m

g pr

ot.

CPE – + – + – +

†

⁎⁎⁎

†

†

†⁎

⁎

(b)

Figure 5: Effect of cocoa polyphenol extract on mitochondrial complex I and complexes II + III in macrophage phenotypes. Macrophage-differentiated THP-1 (M0) cells and polarized (M1, M2) cells were incubated in the absence (−) and in the presence (+) of cocoapolyphenolic extract (CPE) at concentration 100 μM GAE as described under the Materials and Methods. The activities of complex I (a),rotenone sensitive, and complexes II + III (b), antimycin A sensitive, estimated in M0, M1, and M2 phenotypes, through polarograficassay, were expressed as nmol O2/mg total proteins. For further details, see under the Materials and Methods. Error bars represent datafrom 3–5 independent experiments: two-way repeated measures ANOVA (two-factor repetition), followed by Student-Newman-Keulsmethod. ∗p < 0 05, ∗∗∗p < 0 001, cells treated with CPE versus untreated. Comparisons M1 versus M2 within treatment: †p < 0 001.

0

20

40

60

80

100

120

M0 M1 M2

nmol

ATP

/106 ce

lls(%

ctrl)

⁎⁎⁎

†⁎

⁎⁎⁎†

⁎

⁎⁎⁎ ⁎⁎⁎

†

†AaR

AaR

AaR

(a)

0

20

40

60

80

100

120

M0 M1 M2

nmol

ATP

/106 ce

lls(%

ctrl)

⁎⁎⁎†

⁎⁎⁎

†

⁎⁎⁎ ⁎⁎⁎†

†

⁎⁎⁎ ⁎⁎⁎

AaR AaRAaR

(b)

Figure 4: Effect of cocoa polyphenol extract on ATP production by oxidative phosphorylation in macrophage phenotypes. Macrophage-differentiated THP-1 (M0) cells and polarized (M1, M2) cells were incubated in the presence of rotenone (R) and antimycin A (Aa) asindicated under the Materials and Methods. Cellular ATP, expressed as % of control (in the absence of rotenone and antimycin A), wasestimated in the absence (a) and in the presence (b) of cocoa polyphenolic extract at concentration 100 μM GAE. Error bars representdata from 3 independent experiments: two-way repeated measures ANOVA (two-factor repetition), followed by Student-Newman-Keulsmethod. ∗p < 0 05, ∗∗∗p < 0 001, cells treated with R and Aa versus untreated. Comparisons M1 versus M2 within treatment: †p < 0 001.


Measurement of oxygen consumption, performed forevaluating mitochondrial functionality in macrophagephenotypes, in particular, activity of complex I, rotenonesensitive, and complexes II + III, antimycin A sensitive,suggests that M1 macrophages show a lower oxygen con-sumption by mitochondrial respiratory chain. Therefore,M1 cells show a lower respiratory capacity and high levelsof glycolytic ATP. Conversely, the mitochondrial complexesI and II + III consumed more oxygen in M2 macrophages,indicating that these cells have a good respiratory capacity.In the presence of cocoa, oxygen consumption by complexI is similar in M1 and M2 cells. Cocoa treatment has no effecton M0 and M2 macrophages. This suggests that cocoa flavo-noids, due to their antioxidant activity, act mainly at the levelof complex I, which is redox sensitive. Therefore, cocoapolyphenolic extract not only suppresses inflammation inmacrophage inflammatory phenotype but also changesmacrophage metabolism, promoting oxidative pathways.

The main phenolic compounds present in cocoa extractbelong to the flavonoid group, which are powerful antioxi-dants acting on the inflammatory pathway and immunesystem [31, 45]. We demonstrate in this study that cocoaflavonoids, when present together as in the cocoa extract,have a notable anti-inflammatory effect in polarized mac-rophages, as previously demonstrated for other foods, forexample, pomegranate juice [20]. Although the concentra-tion in plasma of cocoa flavonoids and their metabolites isknown to be low in humans [46, 47], research based onflavonoid-enriched cocoa-derived products with enhancedbioavailability is ongoing [48–50]. In this context, it hasbeen reported that, in healthy subjects, (−) epicatechinreached maximal concentrations of 5.92± 0.60μmol/l 2 hafter the consumption of 0.375 g cocoa/kg body wt. of aflavanol-rich cocoa beverage [51]. Furthermore, it mustbe taken into account that macrophages do not differentiatein the circulation and that data from animal modelsreported a tissue and/or dose-dependent accumulation[52, 53]. Therefore, our in vitro results suggest an interestinghypothesis for in vivo study design. The anti-inflammatoryactions of cocoa polyphenols in vitro, which are demon-strated in this study, are likely due to bioavailability offlavonoids present in cocoa, but the exact mechanism bywhich they enter into the cells and the molecular pathwaysinvolved is unclear. There is some evidence that certaincocoa flavonoids can directly interact with cell signallingand gene expression factors, which regulate expression ofmany cytokine genes [54, 55]. Further research is neededto shed light on the interactions between cocoa and cellphysiology, contributing thus to the body of knowledgeof the effects of food compounds on health.

5. Conclusions

The present work demonstrated that cocoa polyphenolicextract is able (i) to reduce inflammatory response in M1macrophage, favoring secretion of anti-inflammatory cyto-kines; (ii) to induce a phenotypic switch in polarized macro-phages, favoring anti-inflammatory or alternative M2-state;(iii) and to influence macrophage metabolism, favoring

oxidative pathways. In this work, we demonstrated theanti-inflammatory and metabolic effects of cocoa and itspolyphenols on polarized macrophages, indicating polarizingability of cocoa toward the M2 phenotype. For using cocoa asdietary supplementation or in the prevention of patholo-gies, more work is needed to better evaluate the effectsof cocoa polyphenolic extract on primary cell lines and/or in vivo and to identify pathways or molecular signalsinvolved in the M1/M2 metabolic switch, induced by thecocoa polyphenols extract.

Abbreviations

Complex I: NADH dehydrogenaseComplex II: Succinate dehydrogenaseComplex III: Cytochrome c reductaseCPE: Cocoa polyphenolic extractGAE: Gallic acid equivalentIFN-γ: Interferon-γIL-1β: Interleukin 1βIL-10: Interleukin 10IL-12: Interleukin 12IL-6: Interleukin 6LPS: LipopolysaccharidePDA: Photodiode arrayPMA: Phorbol 12-myristate 13-acetateRNS: Reactive nitrogen speciesROS: Reactive oxygen speciesTEAC: Trolox equivalent antioxidant capacityTLRs: Toll-like receptorsTNF-α: Tumor necrosis factor-α.

Conflicts of Interest

The authors declare no competing interests.

Authors’ Contributions

Laura Dugo and Maria Giovanna Belluomo equally con-tributed to this work. Mauro Maccarrone and Anna MariaSardanelli are equally senior authors.

Acknowledgments

This work was supported by the local grants of the 2014University of Bari (Italy) and by the “Research and Mobility”project, University of Messina (Italy).

References

[1] B. Bottazzi, A. Doni, C. Garlanda, and A. Mantovani, “Anintegrated view of humoral innate immunity: pentraxins asa paradigm,” Annual Review of Immunology, vol. 28,pp. 157–183, 2010.

[2] P. J. Murray and T. A.Wynn, “Protective and pathogenic func-tions of macrophage subsets,” Nature Reviews Immunology,vol. 11, no. 11, pp. 723–737, 2011.

[3] N. Kamada, T. Hisamatsu, S. Okamoto et al., “Unique CD14intestinal macrophages contribute to the pathogenesis of


Crohn disease via IL-23/IFN-gamma axis,” Journal of ClinicalInvestigation, vol. 118, no. 6, pp. 2269–2280, 2008.

[4] G. K. Hansson and A. Hermansson, “The immune system inatherosclerosis,” Nature Reviews Immunology, vol. 12, no. 3,pp. 204–212, 2011.

[5] S. Gordon, “Alternative activation of macrophages,” NatureReviews Immunology, vol. 3, no. 1, pp. 23–35, 2003.

[6] A. Sica and A. Mantovani, “Macrophage plasticity and polari-zation: in vivo veritas,” Journal of Clinical Investigation,vol. 122, no. 3, pp. 787–795, 2012.

[7] F. O. Martinez, L. Helming, and S. Gordon, “Alternativeactivation of macrophages: an immunologic functionalperspective,” Annual Review of Immunology, vol. 27,pp. 451–483, 2009.

[8] S. Gordon and P. R. Taylor, “Monocyte and macrophageheterogeneity,” Nature Reviews Immunology, vol. 5, no. 12,pp. 953–964, 2005.

[9] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, and A. Sica,“Macrophage polarization: tumor-associated macrophages asa paradigm for polarized M2 mononuclear phagocytes,”Trends in Immunology, vol. 23, no. 11, pp. 549–555, 2002.

[10] T. Lawrence and G. Natoli, “Transcriptional regulation ofmacrophage polarization: enabling diversity with identity,”Nature Reviews Immunology, vol. 11, no. 11, pp. 750–761,2011.

[11] N. Wang, H. Liang, and K. Zen, “Molecular mechanisms thatinfluence the macrophage M1–M2 polarization balance,”Frontiers in Immunology, vol. 5, no. 614, pp. 1–9, 2014.

[12] T. Rőszer, “Understanding the mysterious M2 macrophagethrough activation markers and effector mechanisms,”Mediators of Inflammation, vol. 2015, Article ID 816460,16 pages, 2015.

[13] P. J. Murray, J. E. Allen, S. K. Biswas et al., “Macrophage acti-vation and polarization: nomenclature and experimentalguidelines,” Immunity, vol. 41, no. 1, pp. 14–20, 2014.

[14] D. M. Mosser and J. P. Edwards, “Exploring the full spectrumof macrophage activation,” Nature Reviews Immunology,vol. 8, no. 12, pp. 958–969, 2008.

[15] A. Sica and V. Bronte, “Altered macrophage differentiationand immune dysfunction in tumor development,” Journal ofClinical Investigation, vol. 117, no. 5, pp. 1155–1166, 2007.

[16] C. He and A. B. Carter, “The metabolic prospective and redoxregulation of macrophage polarization,” Journal of Clinical &Cellular Immunology, vol. 6, no. 6, 2015.

[17] D. Duluc, M. Corvaisier, S. Blanchard et al., “Interferon-gamma reverses the immunosuppressive and protumoralproperties and prevents the generation of human tumor-associated macrophages,” International Journal of Cancer,vol. 125, no. 2, pp. 367–373, 2009.

[18] T. Hagemann, T. Lawrence, I. McNeish et al., ““re-educating”tumor-associated macrophages by targeting NF-kappaB,”The Journal of Experimental Medicine, vol. 205, no. 6,pp. 1261–1268, 2008.

[19] A. Saccani, T. Schioppa, C. Porta et al., “p50 nuclear factor-kappaB overexpression in tumor-associated macrophagesinhibits M1 inflammatory responses and antitumor resis-tance,” Cancer Research, vol. 66, no. 23, pp. 11432–11440,2006.

[20] S. Aharoni, Y. Lati, M. Aviram, and B. Fuhrman, “Pome-granate juice polyphenols induce a phenotypic switch in

macrophage polarization favoring a M2 anti-inflammatorystate,” BioFactors, vol. 41, no. 1, pp. 44–51, 2015.

[21] C. Fanali, L. Dugo, G. Tripodo, and L. Santi, “Cocoa polyphe-nols: chemistry, bioavailability and effects on cardiovascularperformance,” Current Medicinal Chemistry, vol. 23, 2016.

[22] S. J. Crozier, A. G. Preston, J. W. Hurst et al., “Cacao seedsare a “super fruit”: a comparative analysis of various fruitpowders and products,” Chemistry Central Journal, vol. 5,p. 5, 2011.

[23] A. Othman, A. Ismail, N. Abdul Ghani, and I. Adenan, “Anti-oxidant capacity and phenolic content of cocoa beans,” FoodChemistry, vol. 100, no. 4, pp. 1523–1530, 2007.

[24] C. Pantano, N. L. Reynaert, A. van der Vliet, and Y. M.Janssen-Heininger, “Redox-sensitive kinases of the nuclearfactor-kappaB signaling pathway,” Antioxidand and RedoxSignal, vol. 8, no. 9-10, pp. 1791–1806, 2006.

[25] E. Ramiro, A. Franch, C. Castellote, C. Andrés-Lacueva, M.Izquierdo-Pulido, and M. Castell, “Effect of Theobromacacao flavonoids on immune activation of a lymphoid cellline,” British Journal of Nutrition, vol. 93, no. 6, pp. 859–866, 2005.

[26] R. di Giuseppe, A. Di Castelnuovo, F. Centritto et al.,“Regular consumption of dark chocolate is associated withlow serum concentrations of C-reactive protein in ahealthy Italian population,” The Journal of Nutrition,vol. 138, no. 10, pp. 1939–1945, 2008.

[27] M. Monagas, N. Khan, C. Andres-Lacueva et al., “Effect ofcocoa powder on the modulation of inflammatory biomarkersin patients at high risk of cardiovascular disease,” TheAmerican Journal of Clinical Nutrition, vol. 90, no. 5,pp. 1144–1150, 2009.

[28] B. Buijsse, E. J. Feskens, F. J. Kok, and D. Kromhout, “Cocoaintake, blood pressure, and cardiovascular mortality: theZutphen Elderly Study,” Archives of Internal Medicine,vol. 166, no. 4, pp. 411–417, 2006.

[29] R. Corti, A. J. Flammer, N. K. Hollenberg, and T. F. Lüscher,“Cocoa and cardiovascular health,” Circulation, vol. 119,no. 10, pp. 1433–1441, 2009.

[30] K. Davison and P. R. C. Howe, “Potential implications of doseand diet for the effects of cocoa flavanols on cardiometabolicfunction,” Journal of Agricultural and Food Chemistry,vol. 63, no. 45, pp. 9942–9947, 2015.

[31] L. Goya, M. Martín, B. Sarriá, S. Ramos, R. Mateos, and L.Bravo, “Effect of cocoa and its flavonoids on biomarkers ofinflammation: studies of cell culture, animals and humans,”Nutrients, vol. 8, no. 4, p. 212, 2016.

[32] T. Perez-Berezo, A. Franch, C. Castellote, M. Castell, andF. J. Pérez-Cano, “Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake,”The Journal of Nutritional Biochemistry, vol. 23, no. 7,pp. 838–844, 2012.

[33] S. Ramos-Romero, F. J. Perez-Cano, E. Ramiro-Puig, A.Franch, and M. Castell, “Cocoa intake attenuates oxidativestress associated with rat adjuvant arthritis,” PharmacologicalResearch, vol. 66, no. 3, pp. 207–212, 2012.

[34] M.Massot-Cladera, A. Franch, F. J. Perez-Cano, andM.Castell,“Cocoa and cocoa fibre differentially modulate IgA and IgMproduction at mucosal sites,” British Journal of Nutrition,vol. 115, no. 9, pp. 1539–1546, 2016.

[35] M. Comalada, D. Camuesco, S. Sierra et al., “In vivo quercitrinanti-inflammatory effect involves release of quercetin, which


inhibits inflammation through down-regulation of the NF-kappaB pathway,” European Journal of Immunology, vol. 35,no. 2, pp. 584–592, 2005.

[36] Y. C. Park, G. Rimbach, C. Saliou, G. Valacchi, and L. Packer,“Activity of monomeric, dimeric, and trimeric flavonoids onNO production, TNF-alpha secretion, and NF-kappaB-dependent gene expression in RAW 264.7 macrophages,”FEBS Letters, vol. 465, no. 2-3, pp. 93–97, 2000.

[37] N. J. Kang, K. W. Lee, D. E. Lee et al., “Cocoa procyanidinssuppress transformation by inhibiting mitogen-activated pro-tein kinase kinase,” The Journal of Biological Chemistry,vol. 283, no. 30, pp. 20664–20673, 2008.

[38] G. Muthian and J. J. Bright, “Quercetin, a flavonoid phytoes-trogen, ameliorates experimental allergic encephalomyelitisby blocking IL-12 signaling through JAK-STAT pathway inT lymphocyte,” Journal of Clinical Immunology, vol. 24,no. 5, pp. 542–552, 2004.

[39] V. L. Singleton, R. Orthofer, and R. M. Lamuela-Raventós,“Analysis of total phenols and other oxidation substratesand antioxidants by means of folin-ciocalteu reagent,”Methods in Enzymology, vol. 299, pp. 152–178, 1999,Academic Press.

[40] C. Fanali, M. G. Belluomo, M. Cirilli et al., “Antioxidantactivity evaluation and HPLC-photodiode array/MS poly-phenols analysis of pomegranate juice from selected Italiancultivars: a comparative study,” Electrophoresis, vol. 37,no. 13, pp. 1947–1955, 2016.

[41] G. Lopez-Castejon, A. Baroja-Mazo, and P. Pelegrin,“Novel macrophage polarization model: from gene expres-sion to identification of new anti-inflammatory molecules,”Cellular and Molecular Life Sciences, vol. 68, no. 18,pp. 3095–3107, 2011.

[42] C. Piccoli, A. Sardanelli, R. Scrima et al., “Mitochondrial respi-ratory dysfunction in familiar parkinsonism associated withPINK1 mutation,” Neurochemical Research, vol. 33, no. 12,pp. 2565–2574, 2008.

[43] C. Pacelli, D. De Rasmo, A. Signorile et al., “Mitochondrialdefect and PGC-1α dysfunction in parkin-associated familialParkinson’s disease,” Biochimica et Biophysyca Acta- Molecu-lar Basis of Disease, vol. 1812, no. 8, pp. 1041–1053, 2011.

[44] A. Carpentieri, E. Cozzoli, M. Scimeca, E. Bonanno, A. M.Sardanelli, and A. Gambacurta, “Differentiation of humanneuroblastoma cells toward the osteogenic lineage bymTOR inhibitor,” Cell Death and Disease, vol. 6, articlee1974, 2015.

[45] F. J. Pérez-Cano and M. Castell, “Flavonoids, inflammationand immune system,” Nutrients, vol. 8, no. 10, 2016.

[46] A. Spadafranca, C. Martinez Conesa, S. Sirini, and G. Testolin,“Effect of dark chocolate on plasma epicatechin levels, DNAresistance to oxidative stress and total antioxidant activity inhealthy subjects,” British Journal of Nutrition, vol. 103, no. 7,pp. 1008–1014, 2010.

[47] M. Urpi-Sarda, M. Monagas, N. Khan et al., “Targeted meta-bolic profiling of phenolics in urine and plasma after regularconsumption of cocoa by liquid chromatography-tandemmass spectrometry,” Journal of Chromatography. A,vol. 1216, no. 43, pp. 7258–7267, 2009.

[48] F. A. Tomas-Barberan, E. Cienfuegos-Jovellanos, A. Marínet al., “A new process to develop a cocoa powder with higherflavonoid monomer content and enhanced bioavailability inhealthy humans,” Journal of Agricultural and Food Chemistry,vol. 55, no. 10, pp. 3926–3935, 2007.

[49] A. Serra, A. Macià, L. Rubió et al., “Distribution of procyani-dins and their metabolites in rat plasma and tissues in relationto ingestion of procyanidin-enriched or procyanidin-richcocoa creams,” European Journal of Clinical Nutrition,vol. 52, no. 3, pp. 1029–1038, 2013.

[50] T. Cifuentes-Gomez, A. Rodriguez-Mateos, I. Gonzalez-Salva-dor, M. E. Alañon, and J. P. Spencer, “Factors affecting theabsorption, metabolism, and excretion of cocoa flavanols inhumans,” Journal of Agricultural and Food Chemistry,vol. 63, no. 35, pp. 7615–7623, 2015.

[51] R. R. Holt, S. A. Lazarus, M. C. Sullards et al., “Procyanidindimer B2 [epicatechin-(4beta-8)-epicatechin] in humanplasma after the consumption of a flavanol-rich cocoa,” TheAmerican Journal of Clinical Nutrition, vol. 76, no. 4,pp. 798–804, 2002.

[52] M. Urpi-Sarda, E. Ramiro-Puig, N. Khan et al., “Distribu-tion of epicatechin metabolites in lymphoid tissues andtestes of young rats with a cocoa-enriched diet,” BritishJournal of Nutrition, vol. 103, no. 10, pp. 1393–1397,2010.

[53] M. Margalef, Z. Pons, F. I. Bravo, B. Muguerza, and A. Arola-Arnal, “Tissue distribution of rat flavanol metabolites at differ-ent doses,” The Journal of Nutritional Biochemistry, vol. 26,no. 10, pp. 987–995, 2015.

[54] J.-E. Kim, J. E. Son, S. K. Jung et al., “Cocoa polyphenols sup-press TNF-α-induced vascular endothelial growth factorexpression by inhibiting phosphoinositide 3-kinase (PI3K)and mitogen-activated protein kinase kinase-1 (MEK1) activi-ties in mouse epidermal cells,” British Journal of Nutrition,vol. 104, no. 7, pp. 957–964, 2010.

[55] E. Ramiro, À. Franch, C. Castellote et al., “Flavonoids fromTheobroma cacao down-regulate inflammatory mediators,”Journal of Agricultural and Food Chemistry, vol. 53, no. 22,pp. 8506–8511, 2005.


Submit your manuscripts athttps://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

effect of cocoa polyphenolic extract on macrophage...

Documents